PROJECT SUMMARY Alzheimer?s disease (AD) is associated with the misfolding of tau and A? proteins. AD shares important molecular characteristics with classical PrP prion diseases ? particularly the misfolding of tau and A? into prions and amyloids. It is known that tau misfolds differently in different tauopathies. A? is conformationally more heterogeneous, although we have seen some correlations with clinical features. It is well established that AD brain homogenates can seed tau and A? misfolding within cells and mouse brains. These models have informed our understanding of disease progression and are widely used to develop therapeutics and imaging agents. However, it is unclear how faithfully they reproduce the structure and activities of the originating patient-derived strains. Given the potential for multiple structural isoforms, it is essential to ask whether these cellular and animal systems reproduce the conformations of tau and A? found in AD patients. Thus, we will determine the fidelity of strain transmission in cellular and animal models. We will examine propagation of a number of patient-derived and in vitro fibrillized conformational strains. Our over-arching hypothesis is that in vitro strains will be less fit, and their conformations will change when passaged in vivo. In contrast, we expect patient-derived amyloids to be more likely to retain their original strains, although this might be influenced by AD genetic factors, including apo? subtypes and TREM2. In Aim 1) we will develop rapid methods to footprint conformational strains. A) Bottom-up and top-down mass spectrometry (MS) methods will be used to identify proteoforms. B) Limited proteolysis will identify structured regions. C) Cross-linking MS will provide coarse-grained information concerning amyloid structures. D) Fluorescence microscopy utilizing multiple fluorescent dyes will distinguish different conformational and structural forms. In Aims 2 and 3) we will determine the fidelity of transmission of tau prions through cellular and animal models. We will passage synthetic and patient-derived tau prions through widely used HEK cell models, cultured neurons, and Tg mice including ones expressing ApoE subtypes and TREM2 mutants. The fingerprints of these strains will be evaluated before and after passage using methods in Aim 1. These studies will elucidate the extent to which host environmental factors influence the transmission of tau and A? prions. Finally, in Aim 4) we will develop structural models of the TREM2-DAP12 signaling complex. TREM2 is a membrane protein involved in microglial A? sequestration and processing. Its extracellular domain interacts directly with amyloid surfaces, while its transmembrane domain binds to the transmembrane domain of its signaling partner, DAP12. With Sali (Project 4) we are developing an integrative model for the signaling complex, which will help elucidate the mechanism of microglial A? processing, providing potential new targets for therapeutic intervention of AD.